Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3.

Peipei Yang,Quan He,Jonathan A Fletcher,Yeqing Wu,Weicai Chen,Yuehong Wu,Grant Eilers,Xuhui Li,Wei Yu,Lili Liu,Wen-Bin Ou

doi:10.18632/oncotarget.8999

Peipei Yang, Quan He + Show 9 more

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https://doi.org/10.18632/oncotarget.8999

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Abstract

The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

Highlights

The human homolog of mouse double minute 2 (MDM2) is an oncoprotein overexpressed in different types of malignant cancers, due to gene amplification or the SNP309 polymorphism in the promoter region of MDM2 [1, 2]
We evaluate the expression of MDM2-full length (FL), MDM2-A and cyclin D1 in different cancer cell lines
Expression of MDM2 full length (FL) and alternatively spliced variant MDM2-A was evaluated by immunoblotting in a group of cancer cell lines including GIST cell lines (GIST882, GIST-T1, and GIST430), breast cancer cell lines (SKOV3, OVCA429, and ES2), non-small cell lung cancer cell lines (PC-9 and A549), mesothelioma cell lines (MESO924, MESO428, and JMN1B), and liposarcoma cell lines (LPS510, LPS141, and LPS141/239) (Figure 1)

Summary

Introduction

The human homolog of mouse double minute 2 (MDM2) is an oncoprotein overexpressed in different types of malignant cancers, due to gene amplification or the SNP309 polymorphism in the promoter region of MDM2 [1, 2]. MDM2 binds p53 at its N-terminal transactivation domain, blocking p53 transcriptional function, while simultaneously, through the C-terminal RING-finger domain, promoting its polyubiquitination and proteasomedependent degradation [7]. P53 enhances MDM2 transcription through p53-specific response elements in the promoter region of MDM2, forming an autoregulatory feedback loop critical to controlling the balance of p53 and MDM2 [8]. Nutlin-3, a specific small-molecule inhibitor of MDM2, blocks the binding of MDM2 with wild type p53, activating the anticancer activity of p53 [9,10,11]. Previous reports have confirmed that Nutlin-3 inhibits cell viability in cancer cell lines harboring wild-type p53, with an attenuated effect in cell lines harboring mutant p53, indicating that p53 mutation is a strong negative predictor of response [11, 12]. Many wild-type p53 cell lines have shown varying sensitivities to Nutlin-3 [12]

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